ABSTRACT The enteric, intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide, which results in as many as one million deaths. Currently, there is no approved vaccine for the prevention of shigellosis, and increasing drug resistance complicates treatment of the disease. The bacteria are acquired by ingestion of contaminated food or water, and upon reaching the colon, Shigella invade the colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. While much is known regarding the mechanism of pathogenesis, there are still gaps in our knowledge. Progress in closing these gaps has been hampered by the lack of a small animal model that accurately mimics the human disease. Cell culture has been used to study aspects of invasion and replication in the cell, but the use of transformed cells in culture does not provide the same environment as the normal human epithelium. A potential solution to this problem is the development of enteroids derived from human intestinal stem cells as a model for Shigella flexneri infection. In this study, we will use human intestinal enteroid monolayers derived from colon tissue to replicate aspects of the bacterial-host interaction that currently cannot be emulated in cell culture. We will use enteroids to test the current paradigms of S. flexneri invasion, intracellular replication, and spread, in order to determine how S. flexneri responds to its normal host environment. In the first Aim, we will establish the basic parameters for colonoid infection by S. flexneri and test the hypothesis that M-cells are required in the invasion process in human intestine. In Aim 2 we will focus on the dynamics and kinetics of intercellular spread of the bacteria in the enteroid monolayers. One aspect of Shigella infection that is poorly understood is the basis for its tropism for colonic epithelium; in Aim 3 we will use human intestinal enteroids derived from duodenal, jejunal, ileal and colonic tissue to determine whether there is tissue specificity for S. flexneri invasion. This will be critical in designing future experiments to determine the host receptors for S. flexneri and the environmental factors that influence S. flexneri invasion efficiency. Completion of this project will not only allow us to examine basic assumptions about Shigella pathogenesis but will also result in the development of a model system for future studies of the interaction between Shigella and the human epithelium.